title: Validation of the first commercial ELISA for type 2N von Willebrand's disease diagnosis.
abstract: Type 2N von Willebrand's disease (VWD) is characterized by a factor VIII (FVIII) deficiency and a low FVIII/VWF ratio related to a markedly decreased affinity of von Willebrand factor (VWF) to FVIII. Type 2N VWD is diagnosed using assays allowing the measurement of plasma VWF capacity to bind FVIII (VWF:FVIIIB). These assays, crucial in order to distinguish type 2N VWD patients from mild haemophiliacs A and haemophilia A carriers, remain exclusively homemade and limited to laboratories possessing a high level of expertise in VWD. We evaluated the first commercial ELISA (Asserachrom® VWF:FVIIIB; Stago) comparated to a reference method in a multicentric study involving 205 subjects: 60 healthy volunteers, 37 haemophiliacs A, 17 haemophilia A carriers, 37 patients with type 2N VWD, 9 heterozygous carriers for a 2N mutation and 45 patients with miscellaneous other types of VWD (all previously characterized). A diluted plasma sample adjusted to 10 IU dL(-1) of VWF:Ag was incubated with a rabbit antihuman VWF polyclonal antibody. After removing the endogenous FVIII, recombinant FVIII (rFVIII) was added and bound rFVIII was quantified using a peroxydase-conjugated mouse antihuman FVIII monoclonal antibody. The intra-assay and inter-assay reproducibility was satisfactory. In all subgroups, both methods were well correlated. All type 2N VWD patients exhibited a markedly decreased VWF:FVIIIB (lower than 15%) and all heterozygous 2N carriers had a moderately decreased VWF:FVIIIB (between 30% and 65%). All controls (healthy subjects, haemophiliacs A and haemophilia A carriers) had a normal VWF:FVIIIB (higher than 80%) except one healthy volunteer and three haemophiliacs who exhibited a moderately decreased VWF:FVIIIB suggesting a heterozygous status for a 2N mutation. In conclusion, the Asserachrom® VWF:FVIIIB is easy to perform, standardized and accurate for type 2N VWD diagnosis with a 100% sensitivity and specificity.
authors.affiliation: Service d'Hématologie Biologique, Hôpital Antoine Béclère, Clamart Cedex, France. agnes.veyradier@abc.aphp.fr
authors.forename: A
authors.initials: A
authors.lastname: Veyradier
authors.forename: C
authors.initials: C
authors.lastname: Caron
authors.forename: C
authors.initials: C
authors.lastname: Ternisien
authors.forename: M
authors.initials: M
authors.lastname: Wolf
authors.forename: M
authors.initials: M
authors.lastname: Trossaert
authors.forename: E
authors.initials: E
authors.lastname: Fressinaud
authors.forename: J
authors.initials: J
authors.lastname: Goudemand
mesh_terms: D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005169:Factor VIII / Q000378:metabolism*; D006467:Hemophilia A / Q000175:diagnosis; D006801:Humans; D009154:Mutation / Q000235:genetics; D011933:Reagent Kits, Diagnostic; D012016:Reference Values; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D056728:von Willebrand Disease, Type 2 / Q000175:diagnosis* / Q000235:genetics; D014842:von Willebrand Diseases / Q000175:diagnosis; D014841:von Willebrand Factor / Q000378:metabolism*
chemical_list: D011933:Reagent Kits, Diagnostic; D014841:von Willebrand Factor; D005169:Factor VIII